During the development of muscle, contractile proteins are first detected in the multinucleated cells formed by the fusion of single myoblasts. The nuclei of these differentiating muscle cells no longer synthesize DNA. In culture, the time of initiation of fusion is controlled by diffusion-mediated processing of the medium which also retards cell proliferation. Although the myoblasts continue to synthesize DNA, the distribution of generation times is more variable due to protraction of G1. We have shown that cells which fuse do spend more time in G1 than cells still in the cycle, but that the distributions of these values overlap. Although it has been suggested, by others, that withdrawal from the cell cycle is an obligatory prerequisite for myogenic differentiation there is no hard data supporting the premise. Using two markers of muscle differentiation, myosin synthesis and the synthesis of the M subunit of CPK, experiments are planned which should detect the presence of ultramicro quantities of these proteins in synchronized myoblasts populations. Combining both immunological and microenzymological techniques with radioautography should allow us to determine whether trace quantities of either or both of the two proteins are synthesized late in G1 in slowly cycling cells or only in cells which can no longer synthesize DNA. During the current grant year it has been established that CPK activity in fusion-blocked (low Ca ions medium) myoblasts reaches levels (per nucleus) comparable to the activity found in multinucleated fibers. Column separation techniques will be employed to determine whether or not the 100-fold increase in activity represents synthesis of the muscle-specific M subunit of CPK. The sequence of regeneration of single, tadpole muscle fibers in vivo has now been elucidated. Using techniques developed in our laboratory the cytoplasm of individual fibers will be labeled to see whether or not label can be found in the regenerating myoblasts after injury. BIBLIOGRAPHIC REFERENCES: Konigsberg, I.R. (1977) The role of the environment in the control of myogenesis in vitro. In: Pathogenesis of the Human Muscular Dystrophies; 5th International Scientific Conference, Muscular Dystrophy Assoc. (ed. L.P. Rowland) Elsevier Press, Amsterdam.